Addition of RTX to washed blood cells from CLL patients, reconstituted in NHS, leads to deposition of C3b(i).

(A-F) The blood from 3 patients (panels A-B; C-D; E-F) was tested. Lymphocytes were identified based on PE CD45 and side scattering (panels A-B; E-F) or PE antihuman IgM and side scattering (panels C-D). The left panels (A,C,E) present the results of addition of RTX, followed by an incubation for 30 minutes (panels A,E) or 5 minutes (panel C) at 37°C. Samples were washed before probing. In panels B and D, RTX was omitted. In panel F, RTX was added to cells in serum-EDTA (to block C activation). (G-J) Samples from the third patient (panels E-F) were analyzed by fluorescence microscopy after probing with either Al488 HB43 and Al 594 mAb 3E7 (panels G-H) or Alexa 594 mAb HB43 and Alexa 488 mAb 3E7 (panels I-J). Both probing schemes reveal a substantial degree of colocalization of the probes. The Alexa 488 and Alexa 594 signals were visualized with the FITC and Texas Red filter, respectively. Original magnification × 100.